Oligonucleotide probes have been used for many years in a variety of different in vivo and in vitro diagnostic assays, and as research tools. In assays, the probes have been employed, for example, as detection agents where an oligonucleotide sequence of interest is linked to a detectable label, for example, an enzyme, fluorophore, radiolabel, or other reporter group. In addition, the oligonucleotide probes can be attached to a support, for example, a particle or solid surface, for the purpose of capturing and/or sorting of nucleic acids. In addition, the oligonucleotide probes can be used as primers or modulators of amplification, ligation, and other enzymatically catalyzed reactions.
Diagnostic assays, for example, diagnostic assays for detecting the presence of a target nucleic acid in a test sample, typically fall into one of two general categories. In one category, the target nucleic acid is amplified either directly or indirectly to produce copies of the target or of a target surrogate, either of which can be detected by any number of methods. Examples of target amplification methods include polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RT-PCR), transcription mediated amplification (TMA), rolling circle amplification (RCA), and ligase chain reaction (LCR), among others. In the other category, the target nucleic acid is not amplified. Rather, the target is detected directly, typically using one or more hybridization probes. There are many formats for this type of assay, which include, for example, a hybridization probe labeled directly (covalently) or indirectly with a reporter group, or solid-phase such as a bead that may be detected is some fashion.
In general, assays where the target is amplified, for example, by PCR, are considered to be both more specific and more sensitive than direct detection assays. However, direct assays generally are considered to be simpler to perform, and often without the need of sophisticated instrumentation. While simplicity can make direct assays more attractive there is a need to improve their sensitivity. The sensitivity of direct assays can be limited because each complementary target nucleic acid only binds to a single labeled probe. This limitation has been addressed using a variety of approaches including, for example, using multiple probes to “paint” the target, using branched or linker-extended probes containing multiple reporter groups, or constructing large “Christmas trees” of probes containing a plurality of reporters (for example, the Urdea b-DNA assay available from Chiron Corporation).
One approach that can be attractive is to have the target nucleic acid (for example, a DNA or RNA molecule) “turnover” a complementary probe or probes that had bound to the target. In this type of assay, the probe turnover or conversion is linked to a detection system. Attempts at constructing such a system have been reported in the literature.
Abe et al. (J. Am. Chem. Soc. (2004) 126: 13980-13986), for example, describe an approach where probe turnover resulted in a 92-fold amplification of the signal in 24 hours. As discussed by Abe et al., a problem that can be experienced when two adjacent probes are ligated either chemically or enzymatically is that the ligated complex invariably is more stable than the starting complex, resulting in “product inhibition.” Essentially, the ligation event makes it even harder to achieve probe turnover. Although Abe et al. attempted to address this problem by selectively destabilizing the ligated product by introducing a several-atom-length flexible linker between the two probes so as to interrupt the two complementary half segments complementary to adjacent positions on the target. It was contemplated that this type of complex would be considerably less favorable entropically than having a direct linkage between the two half probes. However, the 92-fold amplification reported in Abe et al. may not be sufficient to make a significant improvement from the standpoint of applying the system to detection of biologically important targets. It has been contemplated that turnovers of 1,000-fold or greater in 30 minutes may be required for this or another approach to be commercially useful.
Fong et al. (J. Clin. Microbiol. (2000) 38: 2525-2529) describe an assay based on “Cycling Probe Technology” (CBT) which includes a turnover system that is enzymatically modulated. In this assay, a mixed DNA-RNA-DNA probe containing centrally located ribonucleotide linkages hybridizes to a complementary target sequence when the target is present in a sample of interest. The enzyme RNAse H, when added, recognizes the resulting hybrid and cleaves the RNA portion of the duplex. As a result, the probe is cleaved and the resulting cleaved complex dissociates because it is less stable than the starting hybrid. The target sequence then is free to bind another DNA-RNA-DNA probe molecule and the cycle repeats. Labels disposed at the end of the cleaved probe fragments can be captured and/or directly detected.
Dirks et al. (Proc. Natl. Acad. Sci. USA (2004) 101: 15275-15278) describe a “Hybridization Chain Reaction” where two hairpin probes are synthesized in such a way that they are complementary to each other in an overlapping (staggered) fashion. As a result, they hybridize to one another to create a long-duplex DNA. The rate of duplex formation when these hairpins are first mixed is very small because the sequences within the hairpins are trapped in a duplex conformation that renders them unable to bind to each other. Upon addition of a trigger molecule (i.e., a target sequence), hybridization of the trigger molecule to a sticky end of one of the hairpins causes that hairpin to open. The opened hairpin then opens another hairpin, and so on, until all the hairpins are consumed. Thus, prior to trigger addition, the system essentially contains a substantial potential energy that is released by addition of a specific trigger sequence.
It has been recognized that in order for assays to be run outside of a controlled laboratory environment by unskilled personnel, and/or without requiring the use of sophisticated sensitive instrumentation, the assays must be simple but yet sensitive. Typically such assays are developed for point of care diagnostics (genetic and infectious disease testing), detection of bacteria and viruses in the environment, food, water, and other beverages, as well as for biothreat detection. There is a significant need that assays for such applications require little or no sample preparation. As such, the types of complex assays (e.g., PCR and TMA) typically run in clinical or regulated settings such as hospital and reference laboratories, are almost impossible to transition into more demanding uncontrolled settings.
Notwithstanding the foregoing, there is still a need for specific turnover probes that can provide the requisite sensitivity when used in both in vivo and in vitro diagnostic assays.